Isotope-Coded Labeling for Accelerated Protein Interaction Profiling Using MS

Venable, John D.; Steckler, Caitlin; Ou, Weijia; Grünewald, Jan; Agarwalla, Sanjay; Brock, Ansgar

doi:10.1021/acs.analchem.5b01410

Cited by 5 publications

(7 citation statements)

References 52 publications

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“…Thus, the number of TMT peptides was increased from 1 to 10 min and then dropped at 60 min (Figure S1c). The TMT labeling is a rapid reaction, as 1 min of incubation reached almost 20% of TMT peptides (Figure c), consistent with previous work where the labeling time was as short as 1 min for simple protein systems. − To optimize a reasonable handling time for large-scale analysis, we further tested the labeling at a low temperature (on ice) to slow the reaction. We found the reaction rate was too slow at 0 °C compared to 21 °C and could not reach a plateau even with long time incubation (Figure S1d).…”

Section: Resultssupporting

confidence: 83%

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Global Profiling of Lysine Accessibility to Evaluate Protein Structure Changes in Alzheimer’s Disease

Niu

Wang

et al. 2021

J. Am. Soc. Mass Spectrom.

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The linear sequence of amino acids in a protein folds into a 3D structure to execute protein activity and function, but it is still challenging to profile the 3D structure at the proteome scale. Here, we present a method of native protein tandem mass tag (TMT) profiling of Lys accessibility and its application to investigate structural alterations in human brain specimens of Alzheimer's disease (AD). In this method, proteins are extracted under a native condition, labeled by TMT reagents, followed by trypsin digestion and peptide analysis using two-dimensional liquid chromatography and tandem mass spectrometry (LC/LC−MS/MS). The method quantifies Lys labeling efficiency to evaluate its accessibility on the protein surface, which may be affected by protein conformations, protein modifications, and/or other molecular interactions. We systematically optimized the amount of TMT reagents, reaction time, and temperature and then analyzed protein samples under multiple conditions, including different labeling time (5 and 30 min), heat treatment, AD and normal human cases. The experiment profiled 15370 TMT-labeled peptides in 4475 proteins. As expected, the heat treatment led to extensive changes in protein conformations, with 17% of the detected proteome displaying differential labeling. Compared to the normal controls, AD brain showed different Lys accessibility of tau and RNA splicing complexes, which are the hallmarks of AD pathology, as well as proteins involved in transcription, mitochondrial, and synaptic functions. To eliminate the possibility that the observed differential Lys labeling was caused by protein level change, the whole proteome was quantified with standard TMT-LC/LC−MS/MS for normalization. Thus, this native protein TMT method enables the proteome-wide measurement of Lys accessibility, representing a straightforward strategy to explore protein structure in any biological system.

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Section: Resultssupporting

confidence: 83%

“…Moreover, Lys residues are positively charged and usually accessible for chemical labeling. Indeed, applications of Lys-specific labeling reagents to protein structural analysis have been published for purified proteins in small scales. − …”

Section: Introductionmentioning

confidence: 99%

Global Profiling of Lysine Accessibility to Evaluate Protein Structure Changes in Alzheimer’s Disease

Niu

Wang

et al. 2021

J. Am. Soc. Mass Spectrom.

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“…Recently, the amine-reactive quantification reagent, tandem mass tag (TMT), has been used for the same purpose. 53 , 54 Glycine ethyl ester (GEE) in combination with coupling reagents such as carbodiimides is used for modifying carboxyl groups. 55 The more promiscuous reagent, diethyl pyrocarbonate (DEPC) has been shown to modify several amino acid residues (His, Lys, Tyr, Ser, Thr and Cys).…”

Section: Covalent Labelingmentioning

confidence: 99%

“… 71 Lys-specific labeling using the TMT reagent has been applied to antibody–antigen complexes involving therapeutic antibodies. 54 An elegant example of the combination of several labeling methods was reported by the Vachet group. 72 In this work, labeling of lysine residues with NHSA, of arginine residues with 2,3-butanedione and less specific labeling with DEPC have been applied to study the dimerization of β 2 -microglobulin.…”

Section: Covalent Labelingmentioning

confidence: 99%

Cross-linking and other structural proteomics techniques: how chemistry is enabling mass spectrometry applications in structural biology

Leitner

2016

Chem. Sci.

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“…The detailed inspection of crosslinked peptides permits the pinpointing of contact sides between the different proteins. In H/D exchange experiments or by probing proteins by hydroxyl radicals or other reagents (see Section 4.1.3), conformational dynamics due to protein-protein interactions or induced by PTMs, ligand or metal ion bindings may be unravelled (Ramisetty & Washburn, 2011;Venable et al, 2015). Whereas the above approaches are shotgun MS oriented, novel MS methods allow interaction studies to be made on whole protein complexes and other associates (see Section 4.1.5).…”

Section: The Far From Complete Picture Of Interaction Partners Andmentioning

confidence: 99%

Bacterial Electron Transfer Chains Primed by Proteomics

Wessels¹,

Almeida²,

Kartal³

et al. 2016

Advances in Bacterial Electron Transport Systems and Their Regulation

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Electron transport phosphorylation is the central mechanism for most prokaryotic species to harvest energy released in the respiration of their substrates as ATP. Microorganisms have evolved incredible variations on this principle, most of these we perhaps do not know, considering that only a fraction of the microbial richness is known. Besides these variations, microbial species may show substantial versatility in using respiratory systems. In connection herewith, regulatory mechanisms control the expression of these respiratory enzyme systems and their assembly at the translational and posttranslational levels, to optimally accommodate changes in the supply of their energy substrates. Here, we present an overview of methods and techniques from the field of proteomics to explore bacterial electron transfer chains and their regulation at levels ranging from the whole organism down to the Ångstrom scales of protein structures. From the survey of the literature on this subject, it is concluded that proteomics, indeed, has substantially contributed to our comprehending of bacterial respiratory mechanisms, often in elegant combinations with genetic and biochemical approaches. However, we also note that advanced proteomics offers a wealth of opportunities, which have not been exploited at all, or at best underexploited in hypothesis-driving and hypothesis-driven research on bacterial bioenergetics. Examples obtained from the related area of mitochondrial oxidative phosphorylation research, where the application of advanced proteomics is more common, may illustrate these opportunities.

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Isotope-Coded Labeling for Accelerated Protein Interaction Profiling Using MS

Cited by 5 publications

References 52 publications

Global Profiling of Lysine Accessibility to Evaluate Protein Structure Changes in Alzheimer’s Disease

Global Profiling of Lysine Accessibility to Evaluate Protein Structure Changes in Alzheimer’s Disease

Cross-linking and other structural proteomics techniques: how chemistry is enabling mass spectrometry applications in structural biology

Bacterial Electron Transfer Chains Primed by Proteomics

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